Principles & Interpretation of Laboratory Practices in Surgical Pathology Shameem Shariff, Amrit Kaur Kaler
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FixationChapter 1

 
DEFINITION
Fixation is the process by which the constituents of the cells are fixed in a physical and partly chemical state so that they will withstand subsequent treatment with various reagents with minimal loss of significant distortion or decomposition and keep the tissue in as life-like manner as possible.
 
AIMS
  1. Should stop bacterial degeneration, autolysis and putrefaction.
  2. Should not distort the cellular constituents by swelling or shrinkage, and maintain as close a resemblance as possible to the natural structure of tissue components.
  3. The tissue should withstand the chemicals used at various stages of processing.
  4. Allow clear staining of the sections.
  5. To increase tissue consistency to permit the cutting of thin slices of tissue at varying microns.
  6. To increase optical differentiation of cellular structures.
 
QUALITIES OF A GOOD FIXATIVE
An ideal fixative is one, which provides:
  1. Good tissue penetration.
  2. Stabilizes the tissue, preserving the character and distribution of cellular components.
  3. Prevents fixation artifacts.
  4. Prevents structure deformation, maintaining shape and volume (is isotonic).
  5. Preserves cellular constituents.
  6. Safe to handle—nontoxic and nonallergenic.
An easy way to remember is ‘PRISM’, Penetrate tissue easily, Rapid in action, Isotonic, Stable and safe to handle, Minimal loss or damage to physical and chemical composition of tissue and its components. Effects of fixation on tissues include:
  1. 2Hardening of tissues.
  2. Rendering the cells insensitive to hypotonic and hypertonic solutions.
  3. Aiding or inhibiting staining.
 
CLASSIFICATION OF FIXATIVES
 
Based on Components
Fixatives are classified based on the components present as follows:
  1. Simple fixative: Contains a single chemical, e.g. formaldehyde (10% formalin), glutaraldehyde, ethyl alcohol, etc.
  2. Compound fixative: Contains more than one chemical and used as mixtures. Advantage is due to the unequal affinity of each substance for various structural elements:
    1. Formalin based: 10% neutral buffered formalin, 10% neutral buffered formol saline and formol calcium.
    2. Mercurial fixatives: Zenker's solution, Helly's solution and B5 fixative reagent.
    3. Dichromate fixatives: Regaud's solution, Möller's solution and Orth's solution.
    4. Picric acid fixatives: Bouin's solution and Gendre's fluid.
    5. Alcohol-containing fixatives: Carnoy's and acetic alcohol formalin (AAF).
 
Based on the Action on Tissues
Fixatives are classified based on the action on tissues or cells as follows:
  1. Fixatives used in histopathology: They are of two types:
    1. Microanatomical fixatives: Preserves the microscopic structure of the tissue, e.g. formol saline, formol calcium, Zenker's fluid, etc.
    2. Histochemical fixatives: To demonstrate enzymes, e.g. buffered neutral formalin absolute alcohol.
  2. Fixatives used in cytopathology: These are used to preserve intracellular structures. They are divided into two types:
    1. Nuclear fixatives: Carnoy's fluid, Clarke's fluid and Flemming's fluid.
    2. Cytoplasmic fixatives: Champy's fluid and alcohol fixatives.
 
Based on Their Mode of Action
Fixatives are classified based on their mode of action:
  1. Physical methods, e.g. heating, microwaving and freeze drying.
  2. Chemical methods, examples are as follows:
    1. Aldehydes (cross-linking): Formaldehyde, glutaraldehyde and acrolein mercurial fixatives.
    2. Oxidizing agents: Osmium tetroxide and chromate-containing fixatives.
    3. Protein denaturation (coagulants/dehydrants): Acetic acid, methyl alcohol and ethyl alcohol.
3
 
FIXATION METHODS
 
Heat Fixation
Heat fixation is the usual mode of preparing bacteriological smears. It generally preserves overall morphology, but not internal structures. After a smear has dried at room temperature, the slide is gripped by tongs and passed through the flame of a Bunsen burner several times, to ‘heat kill’ and adhere the organism to the slide. This is routinely used with bacteria. Heat denatures the proteolytic enzyme and prevents autolysis. Heat fixation cannot be used in the capsular stain method as heat fixation shrinks or destroys the capsule (glycocalyx), which cannot be seen in stains. It is also used in combination with formal saline.
 
Heat Fixation on Tissues
The tissue is placed in 20–40 mL of fluid (10% formal saline) and heated below the boiling point over the spirit flame for 1 minute or until the tissue floats on the surface. Then it is cooled immediately and the tissue is taken for processing. This method is generally employed fixation before frozen section.
 
Microwave Fixation
Microwave fixation is a well-established technique. It provides better fixation than direct heating. In heat fixation, the energy is absorbed from the outer layer and transferred slowly to the rest of the substance, while microwave provides a homogeneous rise in temperature (controlled heating) and all the molecules take up energy simultaneously by a diffusion process. This overcomes the problem of erratic heating by the direct flame.
 
Principle
Microwave energy interacts with the bipolar molecules resulting in heat denaturation and disulfide bond formation. This process is called microwave stabilization. There is a significant cross-linking of protein molecules with subsequent chemical fixation.
 
Applications
  1. For routine histopathology light microscopy techniques includes staining (standard and special), mucous substance histochemistry, enzyme histochemistry and immunocytochemistry (ICC).
  2. For electron microscopy.
  3. For rapid fixation of routine surgical specimens and especially for processing urgent cardiac and renal biopsies.
  4. For the preparation of botanical and insect material.
4
 
Procedure
The tissue is irradiated directly or irradiated after immersing in formalin solution for a period of 4 minutes, followed by irradiation in buffered formalin for another 4 minutes. Alternatively, the tissue is immersed in formalin for 4 hours, followed by microwave irradiation for 1½ minutes in saline, which gives superior results. The optimal temperature required is 45–55°C. Less heating causes poor sectioning quality, while overheating causes vacuolation of the cytoplasm and nuclear pyknosis.
 
Advantages
  1. It reduces the time for fixation from 12 hours to less than 20 minutes.
  2. There is little difference in the volume changes in tissues fixed by microwaves compared with conventional formaldehyde-fixed material.
  3. Microwave accelerates staining and has no deleterious effect on special staining.
  4. Microwave treated tissues (50°C) postfixed in osmium tetroxide gives good results in electron microscopy.
  5. It is a prerequisite for immunohistochemical staining methods as it stops the extraction of proteins from tissue.
  6. Tissue antigens are better preserved.
 
Freeze Drying and Fixation
Fresh tissues can be frozen with the following:
  • Liquid nitrogen (–190°C)
  • Isopentane cooled by liquid nitrogen (–150°C)
  • Dry ice [solid carbon dioxide (–70°C)]
  • Carbon dioxide gas (–70°C)
  • Aerosol sprays (–50°C).
 
Procedure
The common procedures used in most laboratories are:
  1. Cryostat sections: In this procedure, tissues are hardened by freezing to temperature as low as –30 to –50°C using a coolant in the cryostat, e.g. Freon 22. Small pieces of tissue sent for rapid diagnosis are transferred onto a chuck of the cryostat in a few drops of optimum cutting temperature (OCT) medium. The metal weight in the cryostat is placed on this for a couple of minutes. This helps in fixation. The chuck is then transferred to the cryotome and serial sections are cut and stained.
  2. Quenching procedure: Detailed as follows:
    1. The tissue is cut into thin sections (1 mm thick) and placed in a beaker of isopentane suspended and snap frozen in a flask of liquid nitrogen gas at –150°C (isopentane is an extremely volatile and extremely flammable liquid, at room temperature and pressure). This process is known as quenching. This rapid freezing prevents the formation of 5ice crystals and preserves the tissue. If only liquid nitrogen is used, it forms vapor bubbles around the tissue, thus producing artifacts around the tissue.
    2. The tissue is then transferred to the drying chamber, which is under vacuum and at a higher temperature of –30°C. The ice (tissue water) is removed by sublimation, the water vapor being absorbed by a drying agent such as phosphorus pentoxide. The tissue is impregnated in the embedding medium under reduced pressure as detailed below:
      1. Transfer the dried tissue quickly to a vacuum-embedding medium, i.e. oven containing molten wax.
      2. On sinking to the bottom of the bath, the tissue will be impregnated with wax and takes approximately 10 minutes for complete impregnation.
 
Advantages
  1. Give better preservation of antigenicity by either ICC or immunohistochemistry (IHC).
  2. Not exposed to the organic solvents and therefore minimal chemical alteration of proteins.
  3. Minimizes the denaturation of proteins or inactivation of enzymes and particularly useful for enzyme studies in neuropathology as tissues are processed fresh.
  4. Little shrinkage of tissue.
 
Disadvantages
  • Lacks precise morphological detail
  • Presents a potential biohazard
  • Restricted to research laboratory
  • Formation of ice crystal artifacts.
 
Chemical Fixation
 
Formaldehyde (Aldehydes)
Characteristics
Formaldehyde is commercially available as formalin (40% formaldehyde dissolved in water) and the same is taken as 100% formalin. It is also available as a stable solid form known as paraformaldehyde. Various forms in use are:
  1. 10% formalin: This is most commonly used form in laboratories, which contains:
    1. Formalin (40% of formaldehyde dissolved in water): 10 mL.
    2. Water: 90 mL.
      This in essence is 4% formaldehyde.
  2. 10% formol saline:
    1. Distilled water: 90 mL.
    2. Formalin: 10 mL.
    3. Sodium chloride: 0.9 g.
  3. 6Neutral buffered formalin:
    1. Formalin: 100 mL.
    2. Distilled/Tap water: 900 mL.
    3. Sodium phosphate monobasic monohydrate: 4
    4. Sodium phosphate dibasic anhydrous: 6.5 g.
      It is a widely used fixative (pH should be 7.2–7.4). It is tolerant and prevents the formation of formalin pigment. The tissues can be sectioned even after 1 year. It is also used in IHC.
  4. Formol calcium acetate:
    1. Tap water: 900 mL.
    2. Formalin: 100 mL.
    3. Calcium acetate: 20 g.
      It is good for preserving phospholipids and used for enzyme histochemistry.
Principle
In the first step of fixation, aldehyde groups in formalin form complexes by forming links (methylene bridges) between protein molecules (Fig. 1.1). These methylene bridges subsequently react with several side chains of proteins to form reactive hydroxymethyl side groups. This cross-linkage does not harm the structure of proteins greatly, so that the antigenicity is not lost. However, this cross-linking is reversible by simple washing in water.
Fixation time and temperature
An average tissue requires 10–20 times its volume of formalin/normal buffered formalin for adequate fixation and immersion for 24 hours at room temperature. If the volume of tissues is small a minimum period of 8–12 hours is used. If the temperature is raised to 45°C, fixation time is shortened by 25–40%.
Advantages
  • Easily available and cheap
  • Good penetration and fixation
  • Preservation of fats, myelin, nerve fibers, amyloid and hemosiderin
  • Does not cause excessive tissue hardening.
zoom view
Figure 1.1: Cross-linking of protein molecules by aldehyde groups
7 Disadvantages
  1. It is slow in action (penetration of 1 mm in 1 hour).
  2. On storage it becomes cloudy due to formation of paraformaldehyde, which can be removed by filtration or is inhibited by adding 11–16% methanol in commercial formaldehyde.
  3. Traces of formic acid are formed by oxidation, which decreases the quality of nuclear staining and leaches out hemosiderin resulting in formation of brown-black pigment called formalin pigment, also known as acid hematin. This can be prevented by buffering it with a handful of calcium carbonate. Alternatively, when excess pigment gets deposited as shown in Figures 1.2A and B, it should be removed from those sections.
  4. Unsuitable for the demonstration of fats and enzymes.
  5. It has a denaturing effect on proteins, hence unsuitable for electron microscopy.
  6. Irritation to the eyes/ears and causes dermatitis.
Removal of formalin pigment before staining
Formalin removal is done using the following methods:
  1. Schridde's method: Treat sections for 30 minutes with a mixture of 200 mL of 75% alcohol and 1 mL of 25–28% liquor ammonia. Wash in water.
  2. Verocay's method: Treat sections for 10 minutes with a mixture of 100 mL of 80% alcohol and 1 mL of aqueous potassium hydroxide followed by thorough washing in water.
  3. Kardasewitsch's method: Treat sections for 30 minutes to 1 hour with a mixture of 100 mL of 70% ethyl alcohol and 1 mL of 28% ammonia water. Wash in water.
  4. Lillie's method: Treat sections for 1–5 minutes with a mixture of 50 mL of 75% acetone, 50 mL of 3% hydrogen peroxide and 1 mL of 28% ammonia water; followed by washing in 70% alcohol and then in running water.
  5. Picric acid method: Treat the sections in saturated solutions of picric acid for 5 minutes to 2 hours.
zoom view
Figures 1.2A and B: Formaldehyde fixed staining. A. Low power view; B. Magnified view showing brown-black pigments, i.e. the presence of formalin pigment in the sections. This may hamper interpretation.
8
 
Glutaraldehyde
Characteristics
Glutaraldehyde is commercially available as 25 or 50% stock solution. It is best to use as an acid solution (pH is 3–5) and at a temperature of 4°C. It is amber colored due to presence of impurities such as acrolein, glutaric acid and ethanol. It is purified by adding activated charcoal. Glutaraldehyde is used is concentration of 3–6.5% buffered to pH 7.4 with 0.1 M phosphate or cacodylate.
Principle
Glutaraldehyde is a dialdehyde and stabilizes the protein structure by cross-linkages. This cross-linking is irreversible.
Advantages
  • Better preservation of cellular and plasma proteins than formaldehyde
  • For electron microscopy, as it fixes the tissue rapidly and stabilizes the proteins
  • Better ultrastructural preservation than formaldehyde
  • Fixes the small tissue fragments and needle biopsies (in 2–4 hours at room temperature)
  • Fixation of tissue remains potent for 3 months at 0–4°C
  • Less shrinkage of tissue as compared to formalin
  • More pleasant smell and less irritating to handle.
Disadvantages
  • More expensive
  • Slow penetration, hence tissues must be small
  • Not suitable for carbohydrates, lipids and immunohistochemical methods
  • Secondary fixation with osmium tetroxide may be done for lipids
  • It gives a strong periodic acid-Schiff (PAS) reaction, which can be eliminated by immersing the sections in concentrated glacial acetic acid and aniline oil mixture.
    Note: Other chemicals used in chemical fixation methods are detailed below.
 
TYPES OF FIXATIVES
 
Chemical Fixatives
 
Osmium Tetroxide
Characteristics
Osmium tetroxide is a fixative not routinely used in the laboratory. It is available as a solid form. It is prepared as a 2% stock solution by adding distilled water at a pH of 7.4.
Principle
Osmium tetroxide cross-links with proteins, reacts with unsaturated lipids and forms monoester as well as diester linkages in the tissues. It rapidly fixes the tissue and stains tissue structure due to formation of hexavalent osmium in proportion to the content of reactive and reducing groups.
9 Advantages
  • Suitable for demonstration of lipids
  • Used after postfixation with glutaraldehyde in electron microscopy to retain lipids
  • Good preservation of Golgi bodies and mitochondria
  • Hardens tissue only slightly.
Disadvantages
  • Osmium tetroxide is very expensive
  • Vapors are very harmful to the eyes and throat
  • Causes swelling of the tissues
  • Poor and slow penetration
  • Difficulty in counterstaining after its use.
Flemming's fluid
  • 1% aqueous chromic acid: 15 mL
  • 2% osmium tetroxide: 4 mL
  • Glacial acetic acid: 1 mL.
Flemming's fluid is suitable for electron microscopy work and is a good fixative for myelin in peripheral nerves. It is also a nuclear fixative, but is rarely used because of its poor and uneven penetration. Fixation time is 12–24 hours and should be followed by thorough washing, and storing in 80% alcohol.
 
Mercurials
Mercurials are not commonly used fixatives, but have certain properties good for certain tissues and therefore can be used in such cases. Their main drawback is that several pigments combine with mercury to produce a brownish-black precipitate. These pigments can however be removed by placing sections in iodine solution for 5–10 minutes and then treated with sodium thiosulfate. Mercurial fixatives do not act on lipids.
Zenker's fluid
Compositions of Zenker's fluid are as follows:
  • Distilled water: 100 mL
  • Mercuric chloride: 5 g
  • Potassium dichromate: 2.5 g
  • Sodium sulfate: 1 g
  • Add 5 mL of glacial acetic acid immediately before use.
Zenker's fixatives are recommended for congested tissues, reticuloendothelial tissues including lymph nodes, spleen, thymus and bone marrow. Zenker's fluid fixes nuclei very well and gives good detail. They are also good for trichrome stains. Duration of fixation is 12 hours, smaller tissues (< 3 mm) are fixed in 2–3 hours. The tissue must be washed overnight to remove the excess dichromate and mercuric chloride pigment must be removed with iodine.
Helly's fluid (Spuler's or Maximow's fluid)
Helly's fluid has the same compositions as that of Zenker's fluid, but differs in 5 mL of formalin is added immediately before use instead of acetic acid. 10Zenker formol is slower than Zenker acetate; fixation time is 8–24 hours. It is excellent for bone marrow, spleen, extramedullary hematopoiesis and is recommended for blood-containing organs in general. It preserves cytoplasmic granules and is suitable for Giemsa/Leishman stains.
B5 fixative reagent
  1. Stock reagent A:
    1. Mercuric chloride: 12 g.
    2. Sodium acetate: 2.5 g.
    3. Distilled water: 200 mL.
  2. Stock reagent B:
    1. 10% buffered neutral formalin.
    2. To prepare the working solution, mix 90 mL stock reagent A with 10 mL stock reagent B.
    3. It is used in bone marrows and on lymph nodes, where lymphomas are suspected. Fixation time is 8–12 hours, also used in IHC.
 
Picric Acid Fixatives
Many of these fixatives require a saturated aqueous solution of picric acid. Aqueous picric acid (2.1%) will produce a saturated solution and 5% picric acid is a saturated solution in absolute ethyl alcohol.
Bouin's fluid
  • Picric acid saturated aqueous solution: 75 mL
  • Formalin: 25 mL
  • Glacial acetic acid: 5 mL.
This fixative, which keeps well, penetrates rapidly and evenly, and causes little shrinkage. Tissue fixed in it gives brilliant staining with the trichrome methods. It precipitates protein and forms protein picrates (water soluble), which are yellow in color and can be removed through subsequent changes in 50–70% alcohol. Hence, tissues should not be placed in water directly after fixation. The sections are treated in a saturated solution of lithium carbonate in 70% alcohol for a few minutes or alternatively treat the sections in ethyl alcohol followed by 5% sodium thiosulfate, then wash in running tap water.
It is a good fixative for glycogen as well as connective tissue, hence useful for liver and muscle, but Gendre's fluid is better for this purpose. It is also recommended for fixation of testis, gastrointestinal (GI) tract and endocrine tissue. It gives brilliant staining with Masson's trichrome, Giemsa and Mallory stains. It lysis red blood cells (RBCs) and reduces the amount of demonstrable iron. Lipids are altered. It cannot be used for quantitative studies.
Gendre's fluid
  • Picric acid saturated solution in 95% alcohol: 80 mL
  • Formalin: 15 mL
  • Glacial acetic acid: 5 mL.
    Recommended for carbohydrate fixation.
11 Rossman's fluid
  • Formalin (neutralized): 10 mL
  • Absolute ethyl alcohol saturated with picric acid (approximately 8.5–9%) 90 mL.
    It is similar to Gendre's, but without acetic acid; uses are also similar to the above.
 
Cytological Fixatives
Rapid fixation of smears is necessary to preserve cytological details of cells spread on a glass slide. Fixation means prevention of degeneration of cells and tissue by the autolytic enzymes present in the cells, and preservation of cells as close as possible to the living state. To achieve this, smears are placed in the fixative solutions for specific periods of time before the staining procedure is started. Fixation changes the physical and chemical state of the cells, and determines the subsequent staining reactions that could be carried out on the smears.
 
Properties of Cytological Fixatives
  • Do not excessively shrink or swell cells
  • Do not distort or dissolve cellular components
  • Inactivate enzymes and preserve nuclear details
  • Kill microbes
  • Improve optical differentiation and enhance staining
  • Maintain properties of the tissues and cell components.
 
Wet Fixation
The process of submerging of freshly prepared smears immediately in a liquid fixative is called wet fixation. This is the ideal method for fixing all gynecological and non-gynecological smears. Any of the following alcohols can be used. All alcohol fixatives should be discarded or if necessary for reuse it should be filtered (Whatman No. 1 filter paper) after each use.
12 Ethyl alcohol/Ethanol (95%): The ideal fixative recommended in most of the laboratories for cytological specimen is 95% ethanol. It produces the characteristic effect desired on the nucleus with optimal chromatin detail for cytological preparations. It is a dehydrating fixative. It acts by removing free and bound water thereby changing the structure of proteins so that proteins precipitate, but leave nucleic acids relatively unchanged. It causes cell shrinkage as it replaces water, but only the desired amount of cell contraction. Absolute ethanol (100%): Produces a similar effect on cells, but is much more expensive.
Ether-alcohol mixture: This fixative was originally recommended by Papanicolaou. It consists of equal parts of ether and 95% ethyl alcohol. It is an excellent fixative, but ether is not used in most of the laboratories because of its safety hazards, odor and hygroscopic properties.
Methanol (100%): This is an acceptable substitute for 95% ethanol. Methanol produces less shrinkage than ethanol, but it is more expensive than ethanol. Propanol and isopropanol (80%): Propanol and isopropanol cause slightly more cell shrinkage than ether-ethanol or methanol. By using a lower percentage of these alcohols, the shrinkage is balanced by the swelling effect of water on cells. Hence, 80% propanol is a substitute for 95% ethanol.
Denatured alcohol: It is a form of ethanol that has been changed by the addition of additives in order to render it unsuitable for human consumption. One formula in use is 90 parts of 95% ethanol + 5 parts of 100% methanol + 5 parts of 100% isopropanol.
Time of fixation: Minimum 15–20 minutes of smear fixation prior to staining is essential. Prolonged fixation for several days or even few weeks will not affect the morphology of cells. If smears are to be preserved over a long period of time in alcohol, it is better to store them in capped containers in the refrigerator. Alternatively smears may be removed from the fixative after fixation, wrapped in paper and transported or preserved.
 
Coating Fixative
Coating fixatives are substitutes for wet fixatives. They are aerosols applied by spraying the smear immediately after spreading the cellular contents. They are composed of an alcohol base, which fixes the cells and a wax-like substance, which forms a thin protective coating over the cells, e.g. carbowax (polyethylene glycol) fixative. Diaphine fixative (hairspray) with a high alcohol content and a minimum of lanolin or oil is also an effective fixative. Most of these agents have a dual action in that they fix the cells and on drying forms a thin protective coating over the smear. These fixatives have practical value in situations where smears have to be mailed to another laboratory for evaluation.
The distance from which the slides are sprayed with an aerosol fixative affects the cytological details and 10–12 inches (25–30 cm) is the optimum distance recommended for aerosol fixation. Aerosol sprays are not recommended for bloody smears, because they cause clumping of erythrocytes. Waxes and oils from hairspray (coating) fixatives can be removed, if it is excess. Smears are kept overnight in 95% alcohol for removal of the coating fixative as these may alter staining reactions if they are in excess.
 
Acetone Fixation
Acetone fixation should be short (1 hour) at 4°C and used only on small specimens/smears. Acetone produces excessive shrinkage and hardening, and results in microscopic distortion. It is used for IHC, enzyme studies and in the detection of rabies. It facilitates entry of large molecules as in antibodies for IHC studies.13
 
Alcoholic Fixatives/Dehydrant Fixatives
Alcoholic fixatives/dehydrant fixatives can also be used for tissues. Some of the fluids for this purpose are detailed below.
Clarke's fluid
  • Absolute alcohol: 75 mL
  • Glacial acetic acid: 25 mL.
This solution produces good general histological results for hematoxylin and eosin stains. It preserves nucleic acids, while lipids are extracted. The fixative penetrates rapidly, gives good nuclear fixation and reasonably good preservation of cytoplasmic elements. It is excellent for smears of cell cultures and chromosome analysis.
Carnoy's fixative
  • Absolute alcohol: 60 mL
  • Chloroform: 30 mL
  • Glacial acetic acid: 10 mL.
This is a special purpose fixative for hemorrhagic samples. It penetrates rapidly. The acetic acid in the fixative hemolyzes the RBCs. It is an excellent nuclear fixative as well as preservative for glycogen, but results in considerable shrinkage of cells and tends to produce overstaining with hematoxylin. Overfixing in Carnoy's fluid also results in loss of chromatin material. Carnoy's fixative is also useful for ribonucleic acid (RNA) staining, e.g. methyl green pyronin stain. Carnoy's fixative must be prepared fresh when needed and discarded after each use. It loses its effectiveness on long-standing and chloroform can react with acetic acid to form hydrochloric acid.
Newcomer's fluid
  • Isopropanol/Isopropyl alcohol: 60 mL
  • Propionic acid: 30 mL
  • Petroleum ether: 10 mL
  • Acetone: 10 mL
  • Dioxane: 10 mL.
This fixative also penetrates rapidly and is excellent for the study of chromosomes for which it is better than Carnoy's.
Dichromate fixatives
Time of fixation is 24 hours (critical). The tissues should be washed after fixation and transferred to 70% ethanol; else pigment precipitation occurs. This may cause excessive shrinkage.
Regaud's fluid or Möller's solution
  • Potassium dichromate: 3 g
  • Distilled water: 80 mL
  • At the time of use add 10% formalin: 20 mL.
Regaud's fluid does not keep well for long time and the solutions should only be mixed immediately before use. It penetrates evenly and rapidly, but has a tendency to overharden the tissues. It may be used as a routine fixative, but is particularly good as a cytoplasmic fixative for mitochondria, 14if followed by 4–8 days chromatin in 3% potassium dichromate. Chromaffin tissue is well-demonstrated, but fluids may be improved for this purpose by the addition of 5% acetic acid.
Champy's fluid
  • 3% potassium dichromate: 7 mL
  • 1% chromic acid: 7 mL
  • 2% osmium tetroxide: 4 mL.
The fixative does not keep well for long time and should be freshly prepared each time. It preserves mitochondria and lipids. The tissue must be washed overnight after fixation.
Orth's fluid
  • Potassium dichromate: 2.5 g
  • Sodium sulfate: 1g
  • Distilled water: 90 mL.
    At the time of use add 10 mL of formaldehyde.
Postchromatization is the treatment of tissues with 3% potassium dichromate after normal fixation. It may be carried out either before processing when the tissues are left for 6–8 days in dichromate solutions or after processing when sections before staining are immersed in the dichromate solution for 12–24 hours followed in each case by washing well in running water. This technique is employed to mordant tissues for staining, particularly mitochondria and gives, improved preservation and staining of these elements. Phospholipids are also more resistant to extraction.
 
Dehydrant-cross-linking Fixatives
Compound fixatives with both dehydrant and cross-linking actions include alcohol-formalin mixtures. They produce excellent results in the immunohistochemical identification of specific antigens. In general, most alcohol-based fixatives should be prepared no earlier than 1–2 days before use.
 
Acetic Alcohol Formalin Fixative
  • Formalin: 5 mL
  • Glacial acetic acid: 5 mL
  • 70% alcohol: 90 mL.
    This is the ideal fixative used for cell block preparation of fluid specimens. It is also used for preservation of glycogen.
 
Alcohol Formalin
  • Ethanol 95%: 90 mL
  • Formalin: 10 mL.
If desired 0.5 g of calcium acetate may be added for neutrality. The fixatives denature and precipitate due to disruption of hydrophobic bonds that contribute to the tertiary structure of proteins. It is an excellent fixative for glycogen. It penetrates the tissue rapidly, hence used in preparation of tissue for immunofluorescence.15
 
MAILING OF UNSTAINED SMEARS
Coating fixative such as Carbowax fixative and spray-coating fixative can be used primarily to facilitate transport of smears, mailing, etc.
 
Glycerin Method for Mailing Slides
Smears are first fixed in 95% ethanol for 15 minutes and removed. Two drops of glycerin are placed on smears and covered with a clean glass slide. This may be wrapped in wax paper and mailed to the laboratory in a suitable container.
 
PREFIXATION OF CYTOLOGICAL MATERIAL
Prefixation of cytological material may preserve some specimens for days without deterioration of cells. The most common solutions used for this purpose are:
  1. Ethyl alcohol (50% solution).
  2. Saccomanno's fixative (50% alcohol with 2% Carbowax 1540).
  3. Mucolex (a commercial mucoliquifying preservative for the collection of mucoid and fluid specimens).
Albuminized slides should be used to prepare smears from prefixed samples. Some of the disadvantages of prefixation are precipitation/coagulation of proteins, hardening of cells in spherical shapes and condensation of chromatin. The coagulation of proteins may interfere with the adherence of cells to glass slides. It also ‘rounds up’ the cells; causes the cells to gather together into tight clusters making stain absorption and interpretation difficult.
 
REHYDRATION OF AIR-DRIED SMEARS
Unfixed, air-dried gynecological smears received from peripheral areas can be used for Papanicolaou staining by rehydration method. The simplest rehydration technique is to place air-dried cytological specimens in 50% aqueous solution of glycerin for 3 minutes followed by two rinses in 95% ethyl alcohol and then stained by the routine Papanicolaou method.
 
FACTORS AFFECTING FIXATION
  1. pH and buffer: Normal physiological state of pH must be 6–8. More acidic medium of the fixative makes it less reactive. Acidity also favors the formation of formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphates, bicarbonates, cacodylate and veronal.
  2. Duration of fixation and size of specimen: The thinner the sections (2–3 mm) the better the penetration. There should be a 20:1 ratio of fixative to tissue.
  3. Temperature: Increasing the temperature, as with all chemical reactions will increase the speed of fixation. Hot formalin will fix tissues faster and this is often the first step on an automated tissue processor.
  4. 16 Osmolality: Hypertonic solution gives rise to cell shrinkage, while hypotonic solution causes swelling. About 0.9% of sodium chloride works well.
  5. Agitation: It increases the speed of penetration into the tissues.
  6. Penetration rate: Penetration of tissues depends upon the diffusibility of each individual fixative. It is expressed as:
    d = K√t
    Where,
    d is the depth of penetration;
    K is the coefficient of diffusion (specific for each fixative);
    t is the time.
    For 10% formalin K = 0.78, this means the formalin fixative should not be expected to penetrate more than 1 mm in an hour and it will take approximately 25 hours to penetrate to the center of a 10 mm thick specimen, i.e. 5 mm (= 5² hour). The duration of fixation should be the square of distance the fixative penetrates. Formalin and alcohol penetrate the best, and glutaraldehyde the worst.
 
FIXATION OF INDIVIDUAL TISSUES
  1. Brain: Adequate preautopsy intra-arterial embalming is done. Formol saline is perfused for minimum of 2 weeks via the middle cerebral arteries. Distortion is prevented by suspending the brain in the fluid by a thread. The brain can then be sliced at 1–2 cm interval after fixation.
  2. Eyes: Fixed in the lower compartment of the refrigerator (2–8°C) for 48 hours after the optic nerve is removed. To speed up the fixation, one or two windows are made into the globe after 24 hours.
  3. Renal biopsies: Immunofluorescence biopsies are snap frozen in liquid nitrogen for cryostat sections and then embedded in epoxy resin. Alternatively most laboratories fix the biopsy in an OCT medium in the cryostat itself before sectioning:
    1. For electron microscopy, fixation is done in 2–3% glutaraldehyde.
    2. For paraffin section, neutral buffered formalin.
  4. Gastrointestinal tract specimen: Flexible fiberoptic endoscopies and small biopsies (2–3 mm) in neutral buffered formalin.
  5. Liver biopsies: Core biopsies (2 × 10 mm) fixed in neutral buffered formalin or in alcohol fixative.
  6. Lungs: Infusion with 4% buffered formaldehyde through main bronchi for 1 week, then fixed in neutral buffered formalin.
  7. Lymphoid tissue: Fixed in neutral buffered formal saline or B5.
  8. Muscle: Open small biopsies are fixed in neutral buffered formal saline, while deep frozen for histochemistry.
  9. Testis: Neutral buffered formal saline, Helly's solution and Bouin's fluid gives clear nuclear details, i.e. azoospermia and oligospermia.
  10. Uterus and cervix: Neutral buffered saline.
  11. Bone: Neutral buffered saline.
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SOFTENING HARD TISSUES
Softening is necessary in tissues such as fingernails, hyperkeratotic skin lesions, fibroids, etc. in which the tissue is processed first by washing the tissue in running water overnight, followed by placing the tissue in 4% aqueous phenol for 1–3 days (Lendrum's method).
BIBLIOGRAPHY
  1. Culling CFA. Handbook of Histopathological and Histochemical Techniques, 3rd edition. London: Butterworths;  1976.
  1. JD Bancroft, Gamble M. Theory and Practice of Histological Techniques, 5th edition. Philadelphia: Churchill Livingstone;  2005.
  1. Lynch MJ, Raphael SS. Lynch's Medical Laboratory Technology, 3rd edition. Philadelphia: WB Saunders Company;  1976.
  1. Manual for Cytology, Directorate General of Health Services, Ministry of Health and Family welfare, New Delhi; 2005.
  1. Medawar PB. The rate of penetration of fixatives. J Royal Micros Soc. 1941; 61: 46-57.